Irsch J, Hunzelmann N, Tesch H, Merk H, Maggi E, Ruffilli A, Radbruch A
Institute for Genetics, University of Cologne, Germany.
Immunotechnology. 1995 Aug;1(2):115-25. doi: 10.1016/1380-2933(95)00012-7.
Flow cytometry of the immune system so far has been limited to the analysis of subpopulations according to lineage markers. The cells involved in a particular immune response could not be assayed due to their low frequency. Here we show the potential of antigen-specific high gradient magnetic cell sorting to enrich cells for visualisation in multiparameter cytometry, functional studies and immortalization.
The aim of this study was the development of an efficient technology for staining and isolation of antigen-binding cells from human peripheral blood. In particular, allergen-specific cells from normal and allergic donors should be analysed and compared to develop a cellular diagnosis of allergy.
The rare antigen-specific cells were sorted by high-gradient magnetic cell sorting with MACS. Haptenized phospholipase A2 (PLA2), the major allergen of bee venom, or haptenized ParoI, the major allergenic component of Parietaria officinalis, were used as antigens. The cells from normal and allergic donors, binding to the allergen were characterized phenotypically by immuno-fluorescence. Allergen-specific B-cells were immortalized by EBV transformation.
Allergen-specific cells can be enriched from blood of both allergic and normal donors to purities of up to 75%, by high gradient magnetic cell sorting. The specificity of labelling with allergen was confirmed by establishing allergen-specific EBV-transformed B-cell lines from the sorted cells. Clear differences exist in the cellular composition of allergen-binding cells from normal compared to allergic donors. In normal donors the allergen-binding cells are B-cells expressing CD19 and CD21. In allergic donors, in addition to allergen-binding B-cells, occurring in about equal absolute numbers as in normal donors, basophilic granulocytes are labeled by allergen. These cells express CD38, CD9 and CD25 on their surface, and stain for IgE.
迄今为止，免疫系统的流式细胞术仅限于根据谱系标志物分析亚群。由于参与特定免疫反应的细胞频率较低，无法对其进行检测。在此，我们展示了抗原特异性高梯度磁性细胞分选技术在富集细胞方面的潜力，可用于多参数细胞术可视化、功能研究和永生化。
本研究的目的是开发一种高效技术，用于从人外周血中染色和分离抗原结合细胞。特别地，应分析和比较正常和过敏供体的过敏原特异性细胞，以开发过敏的细胞诊断方法。
通过使用MACS的高梯度磁性细胞分选技术对罕见的抗原特异性细胞进行分选。将半抗原化的磷脂酶A2（PLA2）（蜂毒的主要过敏原）或半抗原化的ParoI（药用墙草的主要过敏原成分）用作抗原。通过免疫荧光对来自正常和过敏供体且与过敏原结合的细胞进行表型特征分析。通过EBV转化使过敏原特异性B细胞永生化。
通过高梯度磁性细胞分选技术，可从过敏和正常供体的血液中富集过敏原特异性细胞，纯度高达75%。通过从分选细胞中建立过敏原特异性EBV转化的B细胞系，证实了过敏原标记的特异性。与过敏供体相比，正常供体的过敏原结合细胞的细胞组成存在明显差异。在正常供体中，过敏原结合细胞是表达CD19和CD21的B细胞。在过敏供体中，除了与正常供体中数量大致相等的过敏原结合B细胞外，嗜碱性粒细胞也被过敏原标记。这些细胞表面表达CD38、CD9和CD25，并可被IgE染色。
